A Phase 3 trial of the RTS,S/AS01E malaria vaccine is underway in multiple African trial sites. A first interim analysis has shown a vaccine efficacy f 55.8% (95% CI 50.6-60.4) against clinical malaria and 47.3% (95% CI 22.4-64.2) against severe malaria. Previous trials have shown that in some circumstances the duration of protection afforded by the vaccine was at least 45 months while in others it waned at 3 months. Initial results of the phase 3 interim analysis have maintained opened questions regarding the duration of vaccine- induced protection, which may be affected by differing malaria transmission intensities among trial sites. We aim to address key gaps in the knowledge of RTS,S mode of action through the analysis of well- characterized plasma and peripheral blood mononuclear cell samples collected in the pediatric phase 3 trial. RTS,S delivered in combination with the AS adjuvant system was designed to elicit strong antibody, TH1 and cytotoxic responses to the circumsporozoite protein (CSP). However, understanding of the mechanisms of protection is only partial. Based on clinical and immunogenicity data from Phase 2 studies, a model of how protection is developed has been proposed. The study hypothesis is that long term immunity against clinical malaria depends on two distinct, but related, mechanisms: 1) initial partial pre-erythrocytic protection via induction of vaccine-specific humoral and cellular immune responses, and 2) long term immunity resulting from enhancement of blood stage immunity facilitated through partial RTS,S protection. Vaccine efficacy may also be affected by parasite-driven immune modulation such as hyporesponsive populations of memory B cells. To define the initial protective immune responses targeting CSP upon RTS,S/AS01E vaccination (Aim 1), we propose to characterize the quality of the antibody response (affinity/avidity, isotypes, functionality) and supporting B- and T helper cells, as well as effector TH1 responses (cytotoxicity, cytokine secretion). To explain RTS,S/AS01E duration of protection by modulation of naturally acquired humoral and cellular immunity (Aim 2), we propose non-biased antibody profiling via protein array with coverage of a large portion of the Plasmodium falciparum proteome, and flow cytometry analysis of asexual blood stage parasite-specific T and B cells associated with the maintenance of long-term protective antibodies. We will use discriminant computational methods to analyze multivariate data and cross-validate predictive signatures of protection. Insights into the immune responses that need to be activated to achieve vaccine efficacy may guide rational design of future vaccines, as these responses could be selectively modulated by appropriately engineering antigens, adjuvants and delivery systems. Identification of immune correlates of protection will also accelerate the evaluation of second generation vaccines against malaria. PUBLIC HEALTH RELEVANCE: RTS,S is the most promising malaria vaccine candidate, currently in advanced stages of testing in African children, although its mode of action remains unclear. The efficacy of RTS,S is moderate, and to develop the next generation of improved vaccines it is critical to understand the immune mechanisms that it induces. We aim to identify antibody and cellular immunological signatures associated with protection elicited by RTS,S with innovative technical and analytical approaches that would allow a deeper and more powerful assessment of vaccine induced immunity.